ABSTRACT While the epitranscriptomic regulation of viral RNA function has begun to attract considerable attention, this work has so far focused entirely on a single epitranscriptomic modification, i.e., the addition of a methyl group to the N6 position of adenosine (m6A). This focus is understandable, given that m6A is the most common epitranscriptomic modification of cellular mRNAs and it has been known for ~40 years that viral transcripts also contain high levels of m6A. Moreover, we and others have reported that m6A regulates HIV-1 replication and we have recently extended this work by showing that m6A also enhances the replication and pathogenicity of influenza A virus (IAV). A second epitranscriptomic modification, the isomerization of uridine to pseudouridine (?), previously thought to be confined to non-coding RNAs, has recently been shown to be almost as prevalent on cellular mRNAs as m6A and we have now observed that the genomic RNAs (gRNAs) packaged into virions of the retrovirus murine leukemia virus display a range of epitranscriptomic modifications, including not only m6A and ? but also 5-methylcytosine (5mC) residues. In this grant application, we propose to use ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to systematically identify and quantitate the epitranscriptomic modifications that are present on highly purified gRNAs isolated from HIV-1 virions. We will then map the precise locations of three of the most prevalent epitranscriptomic modifications on both the gRNA and on HIV-1 mRNAs expressed in CD4+ T-cells and myeloid cells. Next, we will seek to silently mutate these modified sites in the context of an infectious HIV-1 provirus and determine whether, and how, these modifications affect viral gene expression and replication. We will also seek to identify the cellular factors that deposit these epitranscriptomic marks on HIV-1 transcripts and we will then test the effect of loss of expression, or overexpression, of these factors on HIV-1 replication in CD4+ T cells. Finally, we will analyze the effect of environmental stresses, such as heat shock and treatment with drugs of abuse, specifically nicotine and morphine, on the level of epitranscriptomic modification of cellular and viral transcripts expressed in HIV-1 infected cells. In parallel, we will also determine whether these same treatments affect the level of expression of the cellular factors that add or remove epitranscriptomic marks. Together, this research is designed to determine how epitranscriptomic gene regulation modulates HIV-1 replication and begin to shed light on how environmental factors, including drugs of abuse, can affect this process.